L. M. S. Palni

Regulators of Cell Division in Plant Tissues XXIX.* The Activities of Cytokinin Glucosides and Alanine Conjugates in Cytokinin Bioassays

In a number of cytokinin bioassays, the activities of the following compounds were compared: 3-, 7-, and 9-glucosides of 6-benzylaminopurine (BAP); 7- and 9-glucosides of zeatin; O-glucosides of zeatin, dihydrozeatin, and their ribosides; 9-alanine conjugates of zeatin, and BAP. The bioassays included the radish cotyledon, theAmaranthus betacyanin, the oat leaf senescence, and the tobacco pith callus. Cytokinin activity was markedly reduced by 7- and 9-glucosylation in nearly all bioassays, but 3-glucosylation of BAP and O-glucosylation of the zeatin sidechain usually had little effect on activity. However, there were two notable exceptions to this generalization: the activity of O-glucosylzeatin markedly exceeded that of zeatin in the oat leaf senescence assay; 9-glucosyl-BAP and free BAP were similarly active in retarding the senescence of radish leaf discs. The 9-alanine conjugate of zeatin (lupinic acid) and of BAP were markedly less active than zeatin and BAP, respectively, in all bioassays, but the responses evoked by these conjugates at high concentrations in theAmaranthus bioassay approached those caused by the corresponding base. The activities of several new compounds related to the alanine conjugate of BAP were also assessed. To serve as a guide in the selection of the most suitable bioassay for detection of the above-mentioned cytokinin conjugates, the lowest detectable amounts in selected bioassays have been compared.

Detection of cytokinins in a seaweed extract

Abstract trans-Zeatin, trans-zeatin riboside, their dihydro derivatives, isopentenyladenine and isopentenyladenosine have been identified and quantified in Seasol, a commercial extract of Tasmanian Giant Bull kelp, Durvillea potatorum.

Identification of cytokinin glucosides in a seaweed extract

Zeatin-O-glucoside, its dihydro derivative, and dihydrozeatin riboside-O-glucoside have been identified as the main cytokinin-O-glucosides in Seasol, a commercial preparation from Tasmanian giant bull kelp marketed as a liquid organic fertilizer. The analysis, which also indicated the presence of zeatin riboside-O-glucoside, was carried out by gas chromatography-mass spectrometry of the aglucones using the stable isotope dilution method.

6-Benzylaminopurine and its glycosides as naturally occurring cytokinins

Abstract Primary crown gall tumours induced on stems on tomato plants by Agrobacterium tumefaciens yielded extracts with pronounced cytokinin activity. By mass spectrometry and other methods, 6-benzylaminopurine and its 9-β-ribofuranoside were identified as cytokinins in the extracts which also contained a 9-hexoside of 6-benzylaminopurine.

Taxol content in the bark of Himalayan Yew in relation to tree age and sex

Taxol content in the bark of Taxus baccata trees growing in a homogenous (uniform) environment at Jageshwar, District Almora in Central Himalaya has been quantified. The average taxol concentration in the bark of sampled trees was 0.0558+/-0.008% (of dry wt.) and was about 64% higher for male plants (averaged across tree age) in comparison to female trees. Maximum taxol content was recorded in the bark samples collected from trees of >110 yrs age. ANOVA indicates a significant difference in the taxol content of bark from trees of different ages, however, differences were not significant between sexes. Taxol was quantified by HPLC using a standard curve prepared with authentic taxol; the identification of bark taxol was confirmed by UV and mass spectrometry. The total taxol content of the bark of Taxus trees across an age series was found to range between 0.064 to 8.032 g/tree, and a tree of about 100 yrs age can yield 5.74 kg dry bark.

Cytokinin biochemistry in relation to leaf senescence. I: The metabolism of 6-benzylaminopurine and zeatin in oat leaf segments

The metabolism of zeatin and that of 6-benzylaminopurine (BAP) have been compared in oat leaf segments in relation to the markedly differing ability of these cytokinins to retard senescence of such segments. Free BAP and a highly active senescence-retarding metabolite of BAP were detected in oat leaf segments supplied with BAP. The metabolite was identified by mass spectrometry and chromatography as 3-β-D-glucopyranosyl-BAP. The major metabolite of BAP was the 9-glucoside, but this lacked significant senescence-retarding activity. In contrast, in leaf segments supplied with zeatin, no free zeatin and no senescence-retarding metabolite of zeatin were detectable. The major metabolites of zeatin were adenosine, adenine nucleotides, the 9-glucoside, and unidentified polar metabolites. The differing activities of zeatin and BAP in the oat-leaf senescence bioassay appear to be, at least partially, a consequence of their differing metabolism and are not attributable to differences in uptake.

In vitro multiplication of Quercus leucotrichophora and Q. glauca: Important Himalayan oaks

Multiple shoots of Quercus leucotrichophora L. and Q. glauca Thunb. were induced from the intact embryos (decoated seeds) as well as from the cotyledonary nodes (with attached cotyledons but without radicle and primary shoot) of 3-weeks old in vitro grown seedlings on Woody Plant (WP; Lloyd and McCown, 1980) and Murashige and Skoog (MS; 1962) media supplemented with 6-benzyladenine (BA), either alone or in combination with gibberellic acid (GA3)/ indole-3-butyric acid (IBA). BA (22.19 μM) was effective for induction of multiple shoots and addition of GA3 to the medium further enhanced the shoot number and shoot height but resulted in shoot thinness. High frequency shoot multiplication was achieved using cotyledonary nodes. Shoots were further multiplied from the original explant on WP medium supplemented with BA (22.19 μM). Nearly 78% and 67% rooting was obtained in Q. leucotrichophora and Q. glauca microshoots (3–4 cm high), respectively on 1/2 strength WP medium supplemented with IBA (14.76 μM). However, this was associated with basal callus formation. Treatment with IBA (25–100 μM) for 24 or 48 h followed by transfer to PGR free 1/2 strength WP medium not only improved the rooting percentage but also avoided basal callus formation. IBA at 100 μM for 24 h was most effective (90% and 100% rooting in Q. leucotrichophora and Q. glauca, respectively). In vitro rooted plants were hardened and established in garden soil.Growth performance of 6-month-old in vitro raised plants was compared with ex vitro plants (seedlings) of the same age. The photosynthesis and transpiration rates of eight months old in vitro and ex vitro raised plants of both species were measured under different light (0, 600, 900, 1200, 1500 and 2000 μmol m−2s−1) and temperature (20, 25, 30, 35 and 40 °C). Light optimum for photosynthesis was around 2000 μmol m−2s−1 in Q. leucotrichophora and around 1500 μmol m−2s−1 in Q. glauca whereas optimum temperature for photosynthesis was 25 °C in Q. leucotrichophora and 30 °C in Q. glauca. The rate of transpiration at different temperatures (20–40 °C), in the two species, increased with increase in the light intensity up to the highest level, i.e., 2000 μmol m−2s−1. Temperatures beyond 35 °C adversely affected the rate of transpiration in in vitro raised as well as ex vitro plants of both the species. In vitro raised and hardened plants of both the species were comparable to ex vitro plants in terms of gas and water vapour exchange characteristics, within the limits of this study.

Cytokinin Biochemistry in Relation to Leaf Senescence V. Endogenous Cytokinin Levels and Metabolism of Zeatin Riboside in Leaf Discs from Green and Senescent Tobacco (Nicotiana rustica) Leaves

Summary The cytokinin (bases and ribosides) content of leaf discs derived from green and senescent leaves of Nicotiana rustica was analyzed by radioimmunoassay. Zeatin was identified as the major base and dihydrozeatin riboside as the main riboside. Leaf discs from expanded green (presenescent) leaves contained three times higher zeatin levels than those from partly-senescent leaves. The uptake and metabolic fate of [3H]-zeatin riboside supplied to discs from tobacco leaves of different maturities was also investigated. The uptake of zeatin riboside declined with an increase in leaf maturity and senescence. Zeatin riboside was rapidly inactivated mainly by side-chain cleavage. Leaf discs excised from upper green leaves metabolized zeatin riboside into inactive compounds more rapidly than did the basal senescent leaves. It is suggested that higher cytokinin levels in discs from green leaves (in comparison to senescent leaves) may result from higher uptake of xylem cytokinins and/or in situ biosynthesis of cytokinins, rather than a more rapid degradation of zeatin-type cytokinins.

Chemical induction of adventitious root formation in Taxus baccata cuttings

The effect of some auxins (IBA and NAA), phenolic compounds (phloroglucinol, gentisic acid and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin) have been examined for stimulatory effects on adventitious root formation in stem cuttings (current seasons growth) of Taxus baccata L. In general lower concentration (0.25 mM) of both IBA and NAA was more effective in inducing rooting of cuttings taken from both male and female trees. The combined treatment of IBA+NAA (0.25 mM each) showed some success in cuttings from male trees only (55%, compared to 15% rooting in cuttings from female trees). Generally, the callus formation was quite high (≥70%) in all auxin treatments (alone or in combination). Among the phenolics, 40% rooting success was achieved with phloroglucinol only, while coumarin and gentisic acid were ineffective. The combined treatment of auxins and phenolics also failed to promote rooting. On the other hand, Bavistin was extremely effective for callusing (≥90%) as well as rooting (80%). The effectiveness of various compounds tested for rooting of young stem cuttings declined in the order: 0.25 mM IBA>0.05% Bavistin>0.25 mM NAA>1.25 mM IBA>15 mM phloroglucinol>IBA+NAA (0.25 mM each). In addition to the auxins, IBA and NAA that are widely used for commercial propagation, the auxin-like properties of the fungicide Bavistin could be exploited for adventitious rooting in T. baccata, and in other plant species.

Heat-shock induced somatic embryogenesis in callus cultures of gladiolus in the presence of high sucrose

Summary Heat-shock (HS, 50°C 1.h) induced somatic embryogenesis (SE) or shoot bud differentiation (SB) in one year old cytokinin dependent and auxin independent callus cultures of Gladiolus hybridus Hort. grown on high (>174.mM) or low (<116.mM) sucrose medium, respectively. The effect of high sucrose on SE could not be attributed to its role as an osmotic agent alone, because additional sucrose (over and above the normal concentration of 58.mM sucrose) when replaced with mannitol resulted in SB. As in the case of HS, incorporation of putrescine into high sucrose medium also induced SE (and inhibited SB), whereas addition of putrescine to low sucrose medium induced SB. SE was not observed in cultures grown on low sucrose medium. There was more somatic embyro formation in cultures grown on medium gelled with agar than with phytagel. Histological studies revealed that embryo differentiation occurred from the surface layers of callus with no vascular connection with the parent tissue. The somatic embryos could be converted easily into plantlets (ca. 100%) upon transfer to a medium containing less sucrose (58.mM); the plantlets were subsequently transferred to the field, after hardening, with more than 70% survival.